Abstract Understanding how chronic drug exposure alters neuromodulatory circuits such as the dopamine, serotonin, norepinephrine (NE) and neuropeptide systems has been a major focus of addiction research. However, to date it has not been possible to directly examine how prolonged drug exposure alters neural ensemble activity patterns in the neuromodulatory nuclei. To clear this challenge, our CEBRA research will introduce novel imaging techniques, based on the use of a miniature microscope, that will enable neuroscientists to visualize for the first time the neural Ca2+ activity patterns of large sets of individual cells in the neuromodulatory nuclei of freely moving animals. Our approach will also permit in vivo time-lapse imaging of these activity patterns, allowing one to monitor the individual cells over multiple days in a long-term study of drug dependence. We will further increase the potency of the new imaging techniques by combining them with simultaneous optogenetic manipulations of neural activity within the microscope's field-of-view. This combination will allow addiction researchers to observe how a specific input pathway to a neuromodulatory nucleus influences its dynamics. To demonstrate our imaging techniques, we will study the NE neurons in the locus coeruleus (LC). These cells respond to salient cues in the environment and likely have a critical role in mediating physical symptoms of opioid withdrawal. However, knowledge of NE neural activity patterns and how these are changed in drug dependence or withdrawal remains scant. We will use the new imaging techniques to reveal these patterns in normal mice, and mice in states of morphine dependence and withdrawal. We are especially interested in whether drug dependence or withdrawal alters the baseline or sensory-evoked activity patterns of the NE cells, and if the cells' dynamics encodes any of the physical symptoms of drug withdrawal. We will also test whether there are long-lasting changes in NE cell activity patterns that persist even after the physical signs of withdrawal subside. As an initial study using our capacity for optogenetic stimulation concurrent with neural Ca2+ imaging in behaving mice, we will examine how input to the LC from the medial pre-frontal cortex (mPFC) alters the baseline activity patterns of NE cells, as well as their ensemble neural representations of sensory stimuli. Further, we will investigate whether a brain state of morphine dependence or withdrawal alters how the mPFC regulates LC NE neural activity patterns and sensory representations. Overall, our study will provide to the addiction research community important new tools that allow direct visualization of the ensemble dynamics of neuromodulatory neurons in behaving animals, and concurrent means of causally probing the afferent regulation of these activity patterns by using optogenetics. We expect these innovations will have major impact on the field of addiction neurobiology, by allowing researchers to test many longstanding hypotheses about neuromodulatory circuits that could not be previously addressed.